This invention relates to a process for preparing whole blood reference controls having long term stability for devices using electronic means for whole blood determinations, and media therefor.
The importance of a quality control system in the Clinical Hematology Laboratory has been widely emphasized. Especially important is the inclusion of reference blood cell controls which monitor the accuracy and precision of electronic blood cell counting devices. In the past, artificial latex particles and erythrocytes, prepared in fixatives such as glutaraldehyde and formalin, have been used as standard suspensions. These preparations have a number of disadvantages: (1) distortion of shape and volume of the red blood cells; (2) increased viscosity of the suspending media; (3) agglutination of the fixed erythrocytes; (4) inability to perform simultaneous erythrocyte counts and hemoglobin determinations. Several commercial companies have marketed modified whole blood controls for use in monitoring electronic counters, but it is well recognized that there is need for improved stability of reference control material for maintaining the accuracy of red cell counts and other parameters when employing electronic counting methods.
Electronic counters which employ the Coulter principle first described in U.S. Pat. No. 2,656,508 express a true reflection of particle counts. Whole blood reference controls should approximate that of fresh whole blood as closely as possible. Attempts have been made to provide suitably sized particles in stable suspensions by the use of ragweed pollen, polystyrene, latex, various organic materials and tanned red cells. None of these suspensions have proved suitable for use as a standard for red cell counting.
U.S. Pat. No. 3,549,994 to Coulter Electronics, Inc. of Hialeah, Florida describes the Coulter Counter.RTM. Model S, which is a semi-automated analytical instrument that measures seven blood parameters simultaneously; i.e., white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (Hb), Hematocrit (HCT), mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemaglobin concentration (MCHC). Values for the (WBC), (RBC), (Hb), and (MCV) are obtained from direct readings while the (HCT), (MCH), and (MCHC) are computed electronically. The measurement of these seven parameters can be accomplished either on whole blood or on capillary blood. Approximately one milliliter (ml) of whole blood is aspirated (utilized) for the macro measurement; whereas, 44.7 microliters of capillary blood diluted in 10 ml of an isotonic balanced saline solution, or other suitable diluent is used for the micro measurement.
Quality control of the Coulter Counter.RTM. Model S is accomplished with the Coulter Counter.RTM. cell control 4C.RTM., which is a modified whole blood hematology reference control prepared from fresh human blood. It is stable for about 30 days at 2.degree. C. to 8.degree. C., and it is stable for five hours at room temperature before the mean cell volume (MCV) is affected. Cell control 4C.RTM. is not used as a calibration standard. Many workers have used fresh blood from normal persons to calibrate the Coulter Counter.RTM. Model S. Other suitable whole blood reference controls might also be used.
A variety of standard suspensions have been used also to monitor cell counts. One major disadvantage of these suspensions is that, individually, they do not simulate a whole blood sample. For instance, none provide the necessary components for the simultaneous measurements of the seven blood parameters mentioned previously.
The specific parameters of the red blood cells which it is desirable to measure dictate the necessary characteristics of a suitable media for a whole blood reference control. It is desirable to know the volume of the red cell. Once this measurement is ascertained and the red cells have been counted, the packed cell volume or hematocrit (HCT) can be computed. The reference control media also should be capable of equilibrating and stabilizing the volume of red blood cells in the sample so that its cubic volume can be measured (MCV).
The media must be capable of maintaining the chemical and physical integrity of the red blood cells prior to and during the procedure. The blood cells are required to retain the same physical character in the media as exhibited initially. An additional parameter, the red blood cell distribution width (RDW), requires stability of the distribution of the red blood cells. For this purpose the media must be isotonic relative to the solutions in the blood cells.
The Coulter Counter.RTM. Model S Plus, which is capable of electronic sizing and counting of whole blood human platelets which are only 1/2 to 1/3 the diameter of erythrocytes, requires a control such as cell control 4C.RTM., and in addition it requires a control for platelets or materials that simulate human platelets in size and distribution. This has presented a problem in that any particulate matter present in the volume size and distribution range of human platelets will give erroneous results because the presence of foreign particles will result in the enumeration thereof as a blood cell or constituent. High background counts contributed by excess debris cannot be tolerated. Therefore, it is required that the whole blood reference control be rendered free of any particulate matter that would perhaps demonstrate interference in lower size thresholds corresponding to that of human platelet size and distribution. Concomitantly, the media must be bacteriostatic in nature so as to prevent the growth of microorganisms after packaging the media.
New diagnostic parameters utilizing electronic counting devices have made it necessary to find a more stable whole blood reference control. One such parameter is the measurement of red cell distribution width. Normally, under certain defined conditions, such as when fixatives (glutaraldehyde-formaldehyde suspensions) are employed, desired stable red cell distributions are maintained. However, these preparations have a number of disadvantages.